Accurate duplication of chromosomal DNA is vital for faithful transmission of the genome during cell division. However, DNA replication integrity is frequently challenged by genotoxic insults that compromise the progression and stability of replication forks, posing a threat to genome stability. It is becoming clear that the organization of the replisome displays remarkable flexibility in responding to and overcoming a wide spectrum of fork-stalling insults, and that these transactions are dynamically orchestrated and regulated by protein post-translational modifications (PTMs) including ubiquitylation. In this review, we highlight and discuss important recent advances on how ubiquitin-mediated signaling at the replication fork plays a crucial multifaceted role in regulating replisome composition and remodeling its configuration upon replication stress, thereby ensuring high-fidelity duplication of the genome. Replication Stress and Ubiquitin SignalingPrecise and complete replication of cellular DNA during the S phase of each cell cycle is essential for genome stability, cell proliferation, and organismal fitness. The DNA replication process commences prior to S phase, when replication origins are licensed by the loading of inactive double minichromosome maintenance complex (MCM)2-7 hexamers [1-3]. In S phase, origin firing converts these double hexamers into active, bidirectional replication forks, through the recruitment of CDC45 and the GINS complex, leading to formation of the replicative CDC45-MCM2-7-GINS (CMG) helicase that translocates on the leading strand to unwind the duplex DNA template [4,5]. Although eukaryotic DNA replication initiates from multiple replication origins, only a fraction of licensed origins fire during a normal S phase. However, when obstacles that hinder replication fork progression are encountered, activation of otherwise dormant nearby origins provides an important rescue mechanism for completing genome duplication [6]. In S phase, the active replisome, consisting of the CMG helicase, replicative DNA polymerases, the replication factor C (RFC)-loaded clamp proliferating cell nuclear antigen (PCNA), and auxiliary factors, regulates every aspect of bidirectional replication, which occurs continuously on the leading strand and discontinuously on the lagging strand [7].Deregulation of DNA replication, giving rise to replication stress (see Glossary), is a hallmark of cancer cells and a recognized driver of genomic instability [8-10], representing an attractive target for clinical intervention. However, given the sheer size and complex organization of vertebrate genomes, low levels of replication stress occur naturally in most proliferating cells, arising due to obstacles including heterochromatin-imposed barriers (e.g., repetitive DNA sequences and G4 quadruplexes), replication-transcription collisions, ribonucleotide misincorporation, modified or mismatched nucleotides, and helix-distorting adducts that stall the advancing replication machinery [10]. Under normal conditions, such impe...

Receptor-mediated endocytosis is an essential process in signalling pathways for activation of intracellular signalling cascades. One example is the Wnt signalling pathway that seems to depend on endocytosis of the ligand-receptor complex for initiation of Wnt signal transduction. To date, the roles of different endocytic pathways in Wnt signalling, molecular players and the kinetics of the process remain unclear. Here, we monitored endocytosis in Wnt3a and Wnt5a-mediated signalling with membrane capacitance recordings of HEK293 cells. Our measurements revealed a swift and substantial increase in the number of endocytic vesicles. Extracellular Wnt ligands specifically triggered endocytotic activity, which started immediately upon ligand binding and ceased within a period of ten minutes. By using specific inhibitors, we were able to separate Wnt-induced endocytosis into two independent pathways. We demonstrate that canonical Wnt3a is taken up mainly by clathrin-independent endocytosis whereas noncanonical Wnt5a is exclusively regulated via clathrin-mediated endocytosis. Our findings show that membrane capacitance recordings allow the resolution of complex cellular processes in plasma membrane signalling pathways in great detail.

Receptor-mediated endocytosis is an essential process in signaling pathways for an activation of intracellular signaling cascades. One example is the Wnt signaling pathway, which seems to depend on endocytosis of the ligand-receptor complex for initiation of Wnt signal transduction. So far, the role of different endocytic pathways in Wnt signaling, the molecular players and the kinetics of this process are unclear.Here, we monitor endocytosis in Wnt3a and Wnt5a mediated signaling by membrane capacitance recordings of HEK293 cells. Our measurements revealed a fast and substantial increase in the number of endocytic vesicles. This endocytotic activity is specifically elicited by extracellular Wnt ligands; it starts immediately upon ligand binding and ceases over a period of ten minutes. By using specific inhibitors, we can dissect Wnt induced endocytosis into two independent pathways, where canonical Wnt3a is taken up mainly by clathrin-independent endocytosis and Wnt5a exclusively by clathrin-mediated endocytosis.

The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discovered that FAM104A, a protein of unknown function that we named VCF1 (VCP/p97 Cofactor FAM104 1), acts as a novel p97 cofactor in human cells. Detailed structure-function studies revealed that VCF1 directly binds p97 via a conserved novel α-helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.